Abstract
Viral gene delivery is showing great promise for treating retinal disease. Although subretinal vector delivery has mainly been used to date, intravitreal delivery has potential advantages if low retinal transduction efficiency can be overcome. To this end, we investigated the effects of co-injection of glycosaminoglycan-degrading enzymes, singly or in combination, with AAV2 as a method of increasing retinal transduction. Experiments using healthy mice demonstrated that these enzymes enhance retinal transduction. We found that heparinase III produced the greatest individual effect, and this was enhanced further by combination with hyaluronan lyase. In addition, this optimized AAV2-enzyme combination led to a marked improvement in transduction in retinas with advanced retinal degeneration compared with AAV2 alone. Safety studies measuring retinal function by flash electroretinography indicated that retinal function was unaffected in the acute period and at least 12 months after enzyme treatment, whereas pupillometry confirmed that retinal ganglion cell activity was unaffected. Retinal morphology was not altered by the enzyme injection. Collectively these data confirm the efficacy and safety of this intravitreal approach in enhancing retinal transduction efficiency by AAV in rodents. Translating this method into other species, such as non-human primates, or for clinical applications will have challenges and require further studies.
Highlights
Inherited retinal degenerations are a major cause of blindness, affecting approximately 1 in 2,500 people worldwide
By contrast, when AAV2-CAG-GFP was injected in conjunction with the glycosidic enzymes chondroitin ABC lyase (Figure 1C), hyaluronan lyase (Figure 1D; Figure S1B), or heparinase III (Figure 1E; Figure S1C), there was a marked increase in GFP expression in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL), confirming our previous findings.[11]
Quantitative assessment of the transduction efficiency of AAV2CAG-GFP showed a significant increase in the number of GFP+ cell bodies per millimeter of retinal section with addition of the glycosidic enzymes hyaluronan lyase or heparinase III but not with chondroitin ABC lyase (p > 0.05) compared with AAV2 alone (Figure 1K)
Summary
Inherited retinal degenerations (retinal dystrophies) are a major cause of blindness, affecting approximately 1 in 2,500 people worldwide. In most forms, genetic mutations affect the cells in the outer retina—i.e., the photoreceptors and retinal pigment epithelium (RPE)—making these cells primary targets for emerging gene-based therapies. The landmark ocular gene therapy clinical trials for Leber congenital amaurosis 2 (LCA2), a rare form of inherited retinal degeneration,[1,2,3,4,5] have demonstrated the safety and efficacy of delivering therapeutic transgenes via an adeno-associated virus (AAV) vector to the RPE by subretinal injection. In LCA2, the retinal architecture can remain intact for many years,[6] in advanced retinal degeneration, the retina can become thin and fragile, making subretinal delivery of AAV vectors challenging and prone to complications.[5,7,8,9,10] An alternative approach is intravitreal injection, a technically less challenging procedure with a lower risk of complications and, a more broadly applicable approach compared with subretinal injection. Reaching therapeutic levels of transduction in the retina from the vitreous presents challenges and has been a focus of a number of recent preclinical gene therapy studies.[11,12,13,14,15,16,17,18,19,20,21]
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